Human cytokine antibody array kit qah pdd 1

The GCF collected on the papers was diluted with 500 µL of sample diluent provided with the Human Cytokine Antibody Array Kit (QAH-PDD-1, RayBiotech, USA). The protein concentrations of all the samples were measured by the BCA method. To detect the specific cytokine concentrations, all of the samples were diluted again to a concentration of 300 μg/ml. Then, a total of 60 µL of each sample was added to the array. Twenty cytokines, including C-reactive protein (CRP), interferon-γ (IFN-γ), IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, matrix metalloproteinase (MMP)-1, MMP-9, MMP-13, osteoprotegerin (OPG), osteopontin (OPN), osteoactivin (OCN), receptor activator of NF-κB (RANK), transforming growth factor-β1 (TGF-β1), and TNF-α, were detected according to the manufacturer’s instructions.
Briefly, the arrays were incubated with GCF samples for 2 h at room temperature (RT) after blocking. Then, the arrays were washed with PBS three times and incubated with biotin-conjugated antibodies for 2 h at RT. Furthermore, the arrays were incubated with Cy3 equivalent dye-conjugated streptavidin in the dark for 1 h at room temperature after being washed with PBS. Finally, the arrays were visualized with a laser scanner, and the data were analyzed with Axon GenePix software [14 (link)].

GCF samples were eluted from Whatman filter paper in 80 μL of PBS (pH 7.4) containing 0.1 mg/mL phenylmethanesulfonyl fluoride to inhibit the protease activity. Twenty proteins were selected, including CRP, interferon-γ (IFN-γ), IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, macrophage inflammatory protein-1a (MIP-1a), matrix metalloproteinase (MMP)-9, MMP-13, osteoprotegerin (OPG), osteopontin (OPN), osteoactivin, receptor activator of NF-κB (RANK), transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α), all of which have been associated with periodontitis (Garlet, 2010 (link); Shaddox et al., 2011 (link)). Cytokines were detected using a Human Cytokine Antibody Array Kit (QAH-PDD-1, RayBiotech, Norcross, GA, USA), according to the manufacturer's instructions. Briefly, after blocking, the chips were incubated at room temperature (RT) for 2 h with 50 μL of fresh GCF samples. The chips were then washed and incubated for 2 h with biotin-conjugated antibodies at RT. The chips were washed again and incubated for 1 h with Cy3 equivalent dye-conjugated streptavidin in the dark at room temperature. After additional washes, the signals were visualized using a laser scanner, and the data were extracted using Axon GenePix software.